KR101702621B1 - Anti-inflammatory agent containing phlox subulata extract - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising a flower grass extract as an active ingredient. In addition to the absence of cytotoxicity, the flower grass extract showed the transcription level and secretion amount of various cytokine mRNAs related to inflammation. As a result, the ethanol extract can inhibit inflammation most effectively than other solvent extracts, The anti-inflammatory composition comprising the ethanol extract of the present invention as an active ingredient is an anti-inflammatory agent for suppressing inflammation in inflammation-related diseases, a cosmetic composition having anti-inflammatory effect, etc. Can be applied.

Description

ANTI-INFLAMMATORY AGENT CONTAINING PHLOX SUBULATA EXTRACT < RTI ID = 0.0 >

FIELD OF THE INVENTION The present invention relates to a plant extract having anti-inflammatory effect, specifically, a herbal composition containing flower grass extract as an active ingredient.

Inflammation is a localized immune response that minimizes the damage of a cell or tissue when it is damaged or destroyed by various factors, such as harmful substances or organisms from the outside, and restores the damaged area to the original state. And is a useful defense mechanism to remove the products produced by tissue damage. As a result, pain, swelling, redness, fever, or the like may occur as a result of the defensive mechanism, resulting in malfunction. Various factors causing inflammation include physical factors such as trauma, burn, bronze, radioactivity, chemical factors such as acid, and immunological factors due to antibody reaction. In addition, blood vessel and hormone imbalance .

The inflammation normally functions to neutralize or eliminate the causative factor through in vivo inflammatory reaction and regenerate the upper tissue to restore the normal structure and function. However, when the degree of inflammation is over a certain level or becomes chronic, chronic inflammation And the like. In addition to being able to observe inflammatory responses in almost all diseases of clinical diseases, enzymes related to inflammatory reaction play an important role in carcinogenesis.

Recently, it has been known that the progress of the inflammatory reaction in the body is related to COX (cyclooxygenase) enzyme activity. The COX enzyme is a major enzyme involved in the biosynthesis of prostaglandin in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), and two kinds of isozyme enzymes, COX-1 and COX -2 is known to exist. The COX-1 is constantly present in tissues such as the stomach or kidney and is involved in maintaining normal homeostasis, while the COX-2 is involved in mitogen or cytokines in inflammation or other immune responses. Is a transient and rapidly expressed enzyme in the cell.

Another strong inflammatory mediator, nitric oxide (NO), is produced from L-arginine by NO synthase (NOS), and is produced by a variety of substances, such as UV, external stress, endotoxin or cytokines Lt; / RTI > cells. These inflammatory stimuli increase the expression of inducible NOS (iNOS) in the cells, thereby inducing NO production in the cells and activating the macrophages to cause an inflammatory reaction.

Therefore, in order to effectively alleviate inflammation, researches on a substance capable of inhibiting the production of NO are under way. However, some side effects of anti-inflammatory substances developed by these studies are problematic. For example, non-steroidal anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to exhibit side effects such as gastrointestinal disorders by inhibiting COX-1 enzyme as well as inhibiting COX-2 enzyme .

On the other hand, cosmetics used in everyday life are products used for protecting skin and cleansing the shine, but when the cosmetic composition is examined, ingredients different from the purpose of skin protection are used as indispensable substances for product formation. For example, surfactants, preservatives, fragrances, sunscreens, pigments, and other ingredients used to impart other benefits or effects. Ingredients that are essential for the manufacture of cosmetics are generally known to cause skin irritation, rashes, swelling, and various other side effects.

Further, when sebum components, sweat components, and fatty acids, higher alcohols, and proteins in cosmetic components, which are discharged from the body, are decomposed into highly toxic substances by skin-derived bacteria existing on the skin, they may cause skin inflammation It is well known that skin inflammation is caused by ultraviolet rays from the sun.

In this way, the causes of skin adverse effects in cosmetics are always present and various studies have been conducted to solve them. To date, substances that are used for irritation relief and inflammation relief such as erythema and edema include flutenamic acid, ibuprofen, benzydamine, indomethacin, , Steroids such as prednisolone and dexamethasone, and allantoin, azrene, epsilon-aminocaproic acid, hydrocotisone, licorice acid and its derivatives (? -Glycyrrhetinic acid, glycyrrhetinic acid derivative) It is known to be effective for anti-inflammation.

However, the indomethacin generally used as an anti-inflammatory agent is a substance that can not be used in cosmetics. The amount of hydrocotisin is limited. The licorice acid and its derivatives are difficult to stabilize or have poor solubility, It is difficult to achieve a substantial effect. Thus, known anti-inflammatory agents have problems in terms of skin safety and stability in mixing cosmetics, and their use is limited.

Therefore, it is possible to inhibit the production of NO effectively and without any side-effect or cytotoxic effect with a natural-substance-derived substance, so that the anti-inflammatory effect is excellent, and the inflammation can be suppressed without any side effects. The development of materials that can be used in cosmetics is required.

In recent years, in order to meet the needs of consumers, research and development of cosmetics using natural raw materials have been actively conducted.

KR 10-0523753 B KR 10-0530843 B KR 2010-0045575 A KR 2003-0073995 A KR 2002-0001911 A

In order to meet such a demand, the present invention aims to provide a anti-inflammatory composition comprising as an active ingredient a plant extract which is less likely to cause problems related to side effects.

The present invention also provides a plant extract capable of inhibiting inflammation-related NO production and inhibiting inflammatory cytokine, which is safe from the natural plant that can be used for food, unlike the conventional anti-inflammatory substance, And an object of the present invention is to provide an anti-inflammatory agent or a cosmetic composition containing it as an active ingredient.

In order to achieve the above object, the present invention provides a anti-inflammatory composition comprising a flower grass extract as an active ingredient. The flower grass extract may be one obtained by extracting flower grass with an extraction solvent selected from the group consisting of water, an alcohol having 1 to 5 carbon atoms, and a mixture thereof.

The above-mentioned flower grass extract, specifically, a flower grass alcohol extract can effectively inhibit NO secretion, can control various cytokines related to inflammation, is also derived from natural products, has a low possibility of causing side effects , MTT analysis showed no cytotoxicity.

In this respect, the anti-inflammatory composition of the present invention can be provided as an effective ingredient of the anti-inflammatory agent or as an effective ingredient of a composition for treating or preventing inflammatory disease or a composition for suppressing inflammation used for the purpose of inhibiting inflammation.

In order to achieve the above object, the present invention also provides a flower lawn ( Phlox subulata ) as an active ingredient.

In order to achieve the above object, the present invention also provides a flower lawn ( Phlox The present invention provides a composition for treating or preventing an inflammatory disease, comprising an extract of Aspergillus subulata as an active ingredient.

The inflammatory disease may be at least one selected from the group consisting of dermatitis, allergy, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis, arthritis and nephritis.

In order to achieve the above object, the present invention also provides a flower lawn ( Phlox subulata ) as an active ingredient.

The inventors of the present invention have been studying a component derived from a natural substance having an anti-inflammatory effect, and it has been found that a flower grass extract has an anti-inflammatory effect, specifically, a flower grass extract can effectively inhibit the secretion of NO which can directly induce inflammation, NO production and inflammation-inducing cytokine, the present inventors have confirmed that the floral grass extract, especially the floral grass ethanol extract, is excellent in anti-inflammatory effect.

Hereinafter, the present invention will be described in more detail.

The present invention relates to an anti-inflammatory composition comprising a flower grass extract, preferably a flower grass extract, as an active ingredient.

The flower grass ( Phlox subulata ) is a perennial vegetative perennial plant of the foliage of a flowerpot, and is originated in the eastern United States of America, mainly cultivated for ornamental purposes, and also called ground fern. The flowering grass grows in a dry sandy soil and is about 10 cm in height. It grows like a grass with many branches densely growing and covering the ground. The leaves of the flower lawn are opposite to each other, and have a sharp pointed end, a ridged edge, no petiole, and a length of 8 to 20 mm. The flowers of the flower grass bloom from April to September, , Pink or white, one on each of the cracks on the upper part of the stem. The calyx of the flower grass has sharp ends and is divided into 5 pieces and has hairs. The corolla is divided into 5 pieces, the ends are slightly concave, the stamens are 5 pieces, and the fruit is the capsule.

The flower grass extract may be prepared by a conventional method for producing a plant extract. For example, the flower grass extract may be prepared by extracting an extract solvent with leaves, branches, stems, fruits, roots or pulverized products of flower grass, And then fractionating the crude extract with a fraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as an alcohol having 1 to 5 carbon atoms, a diluted alcohol, ethylacetate or acetone, a nonpolar solvent such as ether, chloroform, benzene, hexane or dichloromethane, or a mixture thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, or isopropanol, but is not limited thereto. In addition, the alcohol diluted water may be an alcohol diluted with water at 50% (v / v) to 99.9% (v / v).

The extracting solvent of the flower grass extract of the present invention may preferably be at least one selected from the group consisting of water, an alcohol having 1 to 5 carbon atoms, an alcohol-diluted water, and a mixture thereof, more preferably water, Alcohol, and mixtures thereof, and more preferably, it may be an aqueous solution of ethanol or ethanol. The extraction process may be performed at, for example, 50 ° C to 150 ° C, or 75 ° C to 120 ° C, or 90 ° C to 110 ° C, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 24 hours, or 30 minutes to 12 hours, or 2 hours to 6 hours.

The flower grass extract of the present invention may be prepared according to a conventional method for producing plant extracts. Specifically, it may be a heat extraction method including a hot water extraction method, a cold extraction method, an onion extraction method, an ultrasonic extraction method, A pulverizer or a fractionator may be used.

The extract obtained by the solvent is then subjected to fractionation with any one solvent selected from the group consisting of butanol, hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water and mixtures thereof, preferably butanol Can be further carried out. Two or more kinds of solvents may be used for the fractionation, and each solvent extract may be prepared by sequentially using or mixing the solvents according to the polarity of the solvent

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. The drying can be performed by, for example, freeze drying.

According to one embodiment of the present invention, the ethanol extract of flower grass can effectively inhibit the secretion of NO capable of directly inducing inflammation and effectively inhibit the cytokine associated with inflammation leading to NO secretion and inflammation It is confirmed that it can do.

Accordingly, the anti-inflammatory composition comprising the ethanol extract of flower grass as an active ingredient can be used for suppressing inflammation or for treating, ameliorating or preventing inflammation.

The inflammation may include one or more selected from the group consisting of various dermatitis, allergies, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis, arthritis and nephritis. Allergic rhinitis, allergic rhinitis, anaphylaxis, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, , Food allergies and drug allergies.

Accordingly, the anti-inflammatory composition of the present invention can be applied as a composition for treating or preventing inflammatory diseases or as a food composition for preventing or ameliorating the inflammatory diseases. The food composition may be, for example, a health functional food composition for preventing or ameliorating the inflammatory disease.

The above-mentioned health functional foods are used to control the bio-defense rhythm of the food group or the food composition imparted with added value to function and express the function of the relevant food by physical, biochemical and biotechnological techniques, Means a food which is processed and designed so that the body controlling function of the body is sufficiently expressed to the living body. The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

The health functional food composition for preventing or ameliorating inflammatory diseases according to the present invention may contain 0.001 to 99.9% by weight or 0.01 to 50% by weight or 0.1 to 30% by weight or 0.1% by weight, % To 15% by weight.

The anti-inflammatory composition comprising the flower grass extract as an active ingredient can be directly applied to animals including humans. The animal is a group of organisms corresponding to plants. It mainly consumes organic matter as nutrients, and differentiates digestive organs, excretory or respiratory organs. Preferably, it is a mammal, more preferably a human.

The flower grass extract may be used alone in the anti-inflammatory composition, and may further include a pharmacologically acceptable carrier, excipient, diluent or subcomponent. More specifically, when the composition comprising the flower grass extract is used as a medicine or used for medicinal or pharmaceutical purposes, the flower grass extract may be mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method It can be used diluted with diluent.

In this case, the content of the flower grass extract in the composition may be 0.001% by weight to 99.9% by weight, 0.1% by weight to 99% by weight or 1% by weight to 50% by weight, but is not limited thereto, Depending on the method, the content of the compound may be suitably adjusted in a desired amount.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline. However, the present invention is not limited thereto, and any conventional carrier, excipient or diluent may be used. In addition, the pharmaceutical composition may further comprise at least one selected from the group consisting of conventional fillers, extenders, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, , Fragrances, emulsifiers, preservatives, and the like. The components can be added independently or in combination to the floral grass extract as the active ingredient.

In addition, the composition of the present invention may further comprise a substance used as a known substance having a known anti-inflammatory effect, for example, a substance used as a COX-2 inhibitor, an NO inhibitor, or an anti-inflammatory agent.

When the composition is used as a medicine, it can be administered orally or parenterally. In one example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular.

In addition, the formulations of the compositions may vary depending on the method of use and may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal . In general, solid dosage forms for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, which may contain one or more Excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, Sweeteners, fragrances, preservatives, and the like.

Forms for parenteral administration include creams, looses, ONITMENTS, PLASTERS, LIQUIDS AND SOULTIONS, AEROSOLS, FRUIDEXTRACTS, (ELIXIR), INFUSIONS, SACHET, PATCH or INJECTIONS.

Furthermore, the compositions of the present invention may be formulated using any suitable method known in the art or using methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA .

The dosage of the composition may be determined in consideration of the administration method, the age, sex, the severity of the patient, the condition of the patient, the degree of absorption of the active ingredient in the body, the inactivity and the drug to be used in combination. Kg body weight to 500 mg / kg body weight, 0.1 mg / kg body weight to 400 mg / kg body weight or 1 mg / kg body weight to 300 mg / kg body weight, And may be administered once or several times in divided doses. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides an anti-inflammatory composition comprising the flower grass extract as an active ingredient or an anti-inflammatory agent comprising a flower grass extract as an active ingredient.

The flowering grass extract may preferably be a flowering grass ethanolic extract, more preferably a nonpolar fraction of a flowering grass ethanolic extract.

The anti-inflammatory agent may include the active ingredient alone, and may further include a pharmaceutically acceptable carrier or excipient depending on the formulation, the method of use, and the intended use. When provided as a mixture, the active ingredient may be contained in an amount of 0.1% by weight to 99.9% by weight based on the whole anti-inflammatory agent, but is usually contained in an amount of 0.001% by weight to 50% by weight.

The anti-inflammatory agent is used for the prevention and treatment of various acute inflammatory diseases such as rheumatoid arthritis, lupus, multiple sclerosis such as various chronic inflammatory diseases, septicemia, shock, rejection of organ transplantation, ophthalmic diseases, bronchitis or inflammatory bowel disease It is possible.

Examples of the carrier or the excipient include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gelatin, calcium phosphate, But are not limited to, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Two or more carriers or excipients may be used.

In addition, when the anti-inflammatory agent is weakly formulated, it may further contain conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

In addition, the anti-inflammatory agent of the present invention may further comprise a compound having a known anti-inflammatory activity, or a plant extract, in addition to the active ingredient, and may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the active ingredient.

The formulations of anti-inflammatory agents may be in their preferred forms depending on the method of use, and may be formulated employing methods known in the art to provide rapid, sustained or delayed release of the active ingredient, especially after administration to the mammal. Exemplary formulations include, but are not limited to, excipients, granules, lotions, liniments, rimonadense, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, Suspensions, premixes, infusions, eye drops, tablets, suppositories, injections, main tablets, capsules, creams, pills, soft or hard gelatin capsules.

The anti-inflammatory agent according to the present invention may be used orally or parenterally and may be used, for example, through the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intramuscular, epidural and oral routes, , The age, sex, and weight of the recipient, and the severity of the disease. For example, the anti-inflammatory agent of the present invention can be administered once or more at 0.1 mg / kg (body weight) to 100 mg / kg (body weight) per day based on the active ingredient. However, the above-mentioned dosage is only for illustrative purposes and is not limited to the above range.

The present invention also provides a cosmetic composition comprising an anti-inflammatory composition containing the above-mentioned flower grass extract as an active ingredient or a flower grass extract as an active ingredient.

Since the flower grass extract is derived from a natural substance, it has no problem of side effects, has no cytotoxicity, and is excellent in anti-inflammatory and anti-irritative activity because it has excellent inflammation inhibitory effect. Therefore, And can effectively control the inflammation induced by the external environment. Accordingly, the anti-inflammatory composition comprising the flower grass extract as an active ingredient or the flower grass extract can be used as an effective ingredient of a cosmetic composition having an inflammation improving and alleviating effect.

The flowering grass extract may preferably be a flowering grass ethanolic extract, more preferably a nonpolar fraction of a flowering grass ethanolic extract.

The cosmetic composition may be used as a cosmetic composition for basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, shaving cosmetics or oral cosmetics.

Examples of the cosmetic cosmetic include a cream, a lotion, a pack, a massage cream, and a milky lotion. Examples of the cosmetic cosmetic include a foundation, a makeup base, a lipstick, an eye shadow, an eyeliner, a mascara, an eyebrow pencil, Examples of the cosmetic material include soap, liquid cleansing agent, bathing agent, sunscreen cream, and sun oil. Examples of the cosmetic for hair include shampoo, rinse, hair treatment, hair mousse, hair liquid, pomade, hair color, hair bleach Examples of the cosmetic composition for scalp include hair tonic or scarf treatment, and examples of the cosmetic composition for shaving include aftershave lotion or shaving cream. Examples of the oral cosmetic composition include Toothpaste or mouth washer.

The cosmetic composition may contain, in addition to the active ingredient, a component incorporated in the cosmetic composition, for example, a moisturizer, an ultraviolet absorber, a vitamin, an animal or plant extract, an extinguishing agent, a whitening agent, a vasodilator, an astringent, Hormones and the like may be further added. In addition, the cosmetic composition may further include a medicament or a mechanism necessary for infiltrating or transferring the active ingredient into the dermal tissue.

The formulation of the cosmetic composition may take any appropriate form depending on the intended use and the properties of the cosmetic composition. Examples of the formulation include an aqueous solution, a solubilization system, an emulsion system, an emulsion system, a gel system, a paste system, an ointment system, A two-layer system or a water-oil-powder three-layer system. Examples of the formulations are merely illustrative, and the formulations and forms of the cosmetic composition of the present invention are not limited by the above examples.

The active ingredient may be contained in an amount of 0.001 to 50% by weight, preferably 0.01 to 20% by weight, based on the total weight of the cosmetic composition, , And the content of the active ingredient contained in the present invention is not limited by the above content.

The flower grass extract of the present invention is a natural plant extract and has no side effects or safety problems. As a result of MTT analysis, it has not only cytotoxicity but also the amount of NO associated with inflammation and the transcription of mRNA of various cytokines , It was confirmed that it can effectively inhibit inflammation. Therefore, the anti-inflammatory composition comprising the flower grass extract as an active ingredient is useful as an anti-inflammatory agent for treating or preventing inflammation-related diseases by inhibiting inflammation, a component that is essential for the production of cosmetics, Can be effectively applied to cosmetic compositions and the like that have a characteristic of being able to effectively inhibit inflammation. Therefore, the present invention can be widely used in the medical industry related to the treatment of diseases related to inflammation and in the field of the cosmetic industry related to control of skin inflammation.

FIG. 1 is a graph showing the cytotoxicity of a flower grass ethanol extract according to an embodiment of the present invention. FIG. 1 is a graph showing the cell viability of a flower grass ethanol extract, (vertical axis,%) based on the cell number (cell viability) of the control (control) not treated with the ethanol extract according to the manufacturer's instructions.
FIG. 2 shows the expression of iNOS protein and inhibition of COX-2 protein expression in order to examine the anti-inflammatory effect of ethanol extract of flower grass according to an embodiment of the present invention. In the photograph, '-' indicates no treatment , '+' Means treatment, and the numerical value indicates the throughput (μg / ml) of the ethanol extract of flower grass and the remaining indication indicates the type of treated sample or analyzed protein. FIG. 2A is a photograph showing the result of measurement of the expression amount of iNOS protein, and FIG. 2B is a photograph showing the result of measuring the expression amount of COX-2 protein.
FIG. 3 is a graph showing the results of confirming the inhibition of the production of inflammation-related substances, specifically NO and PGE ₂ , in order to confirm the anti-inflammatory effect of ethanol extract of flower grass according to an embodiment of the present invention. , '-' means no treatment, '+' means treatment, numerical value means throughput (μg / ml) of ethanol extract of flower grass, And the vertical axis of the graph represents the content of the inflammation-related substance to be analyzed. FIG. 3A is a graph showing the results of measuring the content of NO, and FIG. 3B is a graph showing the results of measuring the contents of PGE ₂ .
4 is a graph showing the results of confirming the inhibition of the production of inflammation-related substances, specifically TNF-α and IL-1β, in order to confirm the anti-inflammatory effect of the ethanol extract of flower grass according to an embodiment of the present invention. In the horizontal axis of the graph, '-' means no treatment, '+' means treatment, numerical value means the throughput (μg / ml) of ethanol extract of flowering grass, , And the vertical axis of the graph represents the content of the inflammation-related substance to be analyzed. FIG. 4A is a graph showing the results of measuring the content of TNF-.alpha., And FIG. 4B is a graph showing the results of measuring the content of IL-1beta.

Hereinafter, the structure and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to facilitate understanding of the present invention. However, it should be understood that the following examples are for the purpose of further illustrating the present invention and are not to be construed as limiting the scope of the invention as defined by the appended claims. All technical ideas are to be construed as falling within the scope of the present invention.

< Example  1> Flower grass  Preparation of extract

Flower grass ( Phlox ) Subulata L) was left at room temperature in 50 mL of ethanol for 1 hour, and then subjected to reflux extraction. The extract was concentrated in vacuo to give an extract (3.5 g). In order to conduct cell experiments, extracts were dissolved in DMSO (dimethyl sulfoxide) to prepare samples.

< Example  2> Flower grass  Cytotoxicity test of extract

In order to measure the cytotoxicity of RAW264.7 macrophages of the flower turf ethanol extract of the present invention as a compound of the present invention, 96-well plates (96 wells) were inoculated with 10% heat-inactivated FBS (fetal bovine serum) and penicillin G (100 IU / ml, Gibco co, USA) and streptomycin (100 μg / ml, Gibco co, USA) containing frequency divider in a DMEM culture medium and, 5% CO ₂ incubator (Sanyo, MCO175 ) At 37 占 폚 for 24 hours. Then, the cultured medium (1 × 10 ⁵ cells / ml) was separated into 1-ml 96-well plates, and then 10, 25, 40, 100 and 200 μg / ml of ethanol , And further cultured in a 5% CO ₂ incubator for 24 hours. After the incubation, 50 mg / ml of MTT reagent (M2128, Sigma, USA) was added to the wells and incubated for 4 hours. Then, ELISA reader (Bio-Rad, Microplate reader model 680 ) Was used to measure the absorbance at a wavelength of 570 nm. After repeating the experiment three times each, the average value of the measurement results is shown in Fig. 1 as a relative value to the control group in which no treatment was performed.

As shown in FIG. 1, when the ethanol extract of 200 μg / ml or less of the flower was treated, the cell viability was similar to that of the negative control sample regardless of the concentration of the ethanol extract of the flower grass. From the above results, it was confirmed that the ethanol extract of the flower grass was not cytotoxic up to the concentration of 200 μg / ml and was safe. As shown in FIG. 1, in order to confirm the anti-inflammatory effect of the ethanol extract of the flower lawn described below, the ethanol extract of the flower lawn was treated at a concentration of 200 μg / ml or less.

< Example  3> Flower grass  Extract iNOS  And COX -2 Related anti-inflammatory effects

In order to confirm the anti-inflammatory effect of the ethanol extract of flower grass which was confirmed to be safe because of no cytotoxicity, the following experiment was carried out using RAW264.7 macrophage.

First, the effects of ethanol extracts of flower grass on the expression of enzymes inducing anti-inflammatory effects were investigated in the presence of COX-2 (cyclooxygenase 2), NO (nitric oxide, nitric oxide), which induce the production of PGE ₂ (prostaglandin 2, prostaglandin E2) ) Inducible nitric oxide synthase (iNOS) expression was measured by Western blot analysis. The protein assay method using the Western blot analysis method was carried out as follows.

First, the RAW 264.7 macrophages obtained above were inoculated with 10% heat-inactivated fetal bovine serum (FBS), penicillin G (100 IU / ml, Gibco co, USA) and streptomycin (100 μg / ml, Gibco co, USA) , And cultured at 37 占 폚 for 24 hours using a 5% CO ₂ incubator (Sanyo, MCO175). Thereafter, the cultured culture medium (1 x 10 ⁶ cells / ml) was separated into 1-ml 96-well plates, and then the ethanol extract of flower grass was treated for each concentration, and further cultured in a 5% CO ₂ incubator for 12 hours Lt; / RTI &gt; After the above cultivation, LPS (bacterial lipopoly-saccharide) was treated with 1 μg / ml to induce an inflammatory reaction and further cultured for 24 hours.

After the cultivation, cultured macrophages were collected and centrifuged at 2,000 rpm and 4 ° C for 2 minutes. After centrifugation, the supernatant was removed and the remaining pellet was resuspended in PER-Mammalian protein extraction buffer (protease inhibitor cocktail I, EMD Boisciences, USA) and 1 mM PMSF (phenylmethylsulfonyl fluoride) PER-Mammalian Protein Extraction buffer, Pierce Biotechnology, USA) was added and homogenized. PMSF (0.1 mM PMSF, 5 mg / ml aprotinin, 5 mg / ml pepstatin A and PMSF) were added to 20 mM Tris-HCl buffer (pH 7.4) containing the protein degradation inhibitor and PMSF 1 mg / ml chymostatin). Subsequently, the cytosolic fraction was prepared by centrifuging the homogenized cell liquor at 15,000 × g and 4 ° C. for 10 minutes.

The protein content in the separated fractions was measured using a bicinchoninic acid protein kit (BCA).

Western blot analysis was performed by measuring the expression of iNOS and COX-2 from the cytoplasmic fraction as follows.

First, the protein content contained in the cytoplasmic fractions obtained using the PER-Mammalian protein extraction buffer containing the protein degradation inhibitor and PMSF was measured using a Lowry protein assay kit (P5626, Sigma Chemical Co., USA) Respectively. The protein solution separated by the same amount as above was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoresed on an ECL nitrocellulose membrane (Bio-Rad, USA) Transferred. The protein-transferred ECL nitrocellulose membrane was blocked with 5% skimmed milk, then washed, and the cells were washed with ECL detection system (Amersham Pharmacia Biotech, USA) according to the protocol provided The amount of protein expression was confirmed, and the results are shown in FIG.

Specifically, the antibody reaction according to the ECL detection system was performed by adding an antibody against COX-2 and iNOS (primary antibody, Santa Cruz Biotechnology, USA), reacting at 4 ° C for 24 hours, washing the membrane, (HRP conjugated anti-rabbit antibody) was added, followed by washing for 1 hour, followed by washing and confirming the signal by the antibody.

As shown in FIG. 2, iNOS and COX-2 proteins associated with inflammation were not observed in the LPS-untreated control group, whereas iNOS and COX-2 protein bands were clearly observed in the LPS-only treatment. On the other hand, it was confirmed that the band of iNOS and COX-2 protein was blurred in a concentration-dependent manner when the ethanol extract of the present invention was added to the flower grass despite the LPS treatment.

From the above results, it was confirmed that the addition of the ethanol extract of the flower lawn of the present invention resulted in inhibiting the expression of iNOS and COX-2 and inhibiting the inflammation, thereby having an anti-inflammatory effect.

< Example  4> Identification of anti-inflammatory effect by inhibition of inflammation-related substance formation

In order to confirm the anti-inflammatory effect of the ethanol extract of flower turf confirmed to have the anti-inflammatory effect related to the inhibition of the production of iNOS and COX-2, further inhibition of inflammation-related substances, specifically NO, PGE ₂ , TNF-α and IL-1β The following experiment was carried out.

First, RAW 264.7 macrophages were treated with ethanol extract of flowering grass for 12 hours, treated with 1 μg / ml of LPS and further cultured for 24 hours to induce an inflammatory response Respectively. After the above culture, centrifugation was carried out at 13,000 x g and 4 ° C for 2 minutes, and the supernatant was collected.

The secretion amount of NO in the collected supernatant was measured according to the protocol provided by Promega using Griess reagent system (Promega, USA). Specifically, 100 μl of the supernatant of each control and experimental group and 0.1% (w / v) N- (1-naphathyl) -ethylene diamine in the same amount of Griess reagent (5% Dissolved solution and 1% (w / v) sulfanil amide) were mixed and allowed to react at room temperature, that is, at 15 ° C to 20 ° C for 10 minutes. Then, the content of NO was measured by measuring the absorbance at 525 nm using an ELISA plate reader. The measurement results are shown in Fig. 3A.

The secretion amount of PGE ₂ in the collected supernatant was measured using commercially available PGE ₂ (R & D Systems, USA) according to the protocol provided by the manufacturer. Specifically, the supernatant of each control and test group was added to a 96-well plate coated with antibody against PGE ₂ , followed by addition of a secondary antibody (enzyme-linked polyclonal antibody specific for PGE ₂ ) , And washed to remove unattached secondary antibodies. Then, the content of PGE ₂ was measured by adding the substrate solution and measuring the fluorescence intensity at 450 nm. The measurement results are shown in FIG. 3B.

The amount of secretion of TNF-α and IL-1β in the supernatant was measured using a commercially available assay kit (R & D Systems, USA) according to the manufacturer's protocol. Specifically, the supernatants of the respective control and experimental groups were added to 96-well plates coated with antibodies to mouse-derived TNF-α or IL-1β, respectively, and then incubated with a secondary antibody (enzyme-linked polyclonal antibody specific for mouse TNF-α or IL-1β) was added and allowed to react for 2 hours, followed by washing to remove unattached secondary antibodies. Subsequently, the content of TNF-α or IL-1β was measured by adding the substrate solution and measuring the fluorescence intensity at 450 nm. The measurement results are shown in Fig.

As shown in FIGS. 3A and 3B, in the control group not supplemented with LPS, the NO production amount and the PGE ₂ In the case of addition of LPS sample, the amount of NO production and PGE ₂ concentration were significantly increased. It was confirmed that the production amount was reduced and decreased. The results, iNOS and COX-2 showed a similar trend with protein production inhibition, reducing the NO generation amount in accordance with iNOS Inhibitory therefrom and, and in accordance with the COX-2 protein inhibited PGE ₂ as shown in Example 2 The amount of production was also confirmed again.

In addition, as shown in Figs. 4A and 4B, it was confirmed that the ethanol extract of flower lawn was also able to effectively inhibit the generation of cytokines in a concentration-dependent manner even in inflammation-derived cytokines, and the anti- . Specifically, the expression levels of TNF-α and IL-1β increased by the addition of the LPS sample were decreased in a concentration-dependent manner by the addition of the ethanol extract of flower grass. Especially, in the case of IL-1β, Was found to be superior.

As a result of the MTT analysis, the extract of flower grass which can be used for edible use was confirmed to have no cytotoxicity, and the results related to inflammation caused by NO (NO secretion amount) And the results related to the mRNA transcription amount of various inflammatory cytokines, it was found that the ethanol extract of flower grass has remarkably excellent anti-inflammatory effect as compared with other solvent extracts, Since the ethanol extract of the flower lawn was found to be superior in anti-inflammatory effect to other solvent fractions, the flower lawn extract of the present invention, in particular, the floral lawn ethanol extract of the present invention can be used as a substitute for existing inflammation drugs, It is possible to appropriately control the occurrence of inflammation caused by the cosmetic composition It is expected that it can be used as an active ingredient of

Claims (6)

A pharmaceutical composition for antiinflammation comprising an extract of Phlox subulata as an active ingredient. The method according to claim 1,
Wherein the flower grass extract is obtained by extracting at least one selected from the group consisting of water, an alcohol having 1 to 5 carbon atoms, and a mixture thereof with an extractant. delete A pharmaceutical composition for treating or preventing an inflammatory disease comprising an extract of Phlox subulata as an active ingredient. delete Flower grass ( Phlox subulata ) as an active ingredient.

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